Liquidator 96 Liquid Handling Systems, Ranging from Multichannel Pipettes to Laboratory Auto- Mation Devices

Higher Efficiency in the Lab New Liquidator® 96 Volume Range

Two tools that increase efficiency in scientific research are microtiter plates and liquid handling systems. Microtiter plates allow for the approximation of many samples into a small amount of space. Available with 96, 384 and even more wells, they are convenient because they interface with a wide range of scientific instruments, enabling samples to be quickly analyzed.

Another advantage of microtiter plates is that they can be filled using various

Liquidator 96 liquid handling systems, ranging from multichannel pipettes to laboratory automation devices. These instruments reduce the amount of time required to fill 96- and 384-well plates, but have limitations: • Researchers using multichannel pipettes with microtiter plates risk skipping a row or column of wells within the plate or pipetting twice into the same row or column. • Automated devices remove the element of human error from reliably pipetting into 96-well plates, but these devices are generally quite expensive and require in-depth training to operate as well as dedicated space within the laboratory. METTLER TOLEDO’s Rainin Liquidator 96 is a manual 96-channel pipetting device that allows researchers to accurately and precisely dispense solutions into 96- and 384-well plates. Liquidator obviates the disadvantages of multichannel pipettes and automation systems while capitalizing on their strengths.

Benefits of the Liquidator 96 include: • Two volume ranges – 0.5 to 20 µL and 5 to 200 µL accommodate most scientific applications. • Speed – Liquidator fills 96-well plates in 6 seconds vs. 30 seconds with a 12-channel pipette and 295 seconds using a single-channel pipette. • Accuracy and precision – both Liquidator models have superb accuracy and precision and can be used in applications like qPCR and analytical enzymatic assays. • Reproducibility – Liquidator’s 96 channels aspirate and dispense with very high consistency. Benefits of the Liquidator 96 include: • Two volume ranges – 0.5 to 20 µL and 5 to 200 µL accommodate most scientific applications. • Speed – Liquidator fills 96-well plates in 6 seconds vs. 30 seconds with a 12-channel pipette and 295 seconds using a single-channel pipette. • Accuracy and precision – both Liquidator models have superb accuracy and precision and can be used in applications like qPCR and analytical enzymatic assays. • Reproducibility – Liquidator’s 96 channels aspirate and dispense with very high consistency. • Validated – beyond simple testing, the 0.5 to 20 µL (20 µL Liquidator) and 5 to 200 µL (200 µL Liquidator) models have been used in many real-life laboratory applications including ELISA, PCR, qPCR and enzyme assays. • Fewer errors – unlike single- and multi-channel pipettes, Liquidator pipettes all 96 channels

Liquidator 96 simultaneously, eliminating the possibility of skipping or repeating rows and wells. • Moderate weight and size – Unlike laboratory automation systems, the Liquidator can be moved around the laboratory with relative ease and requires little space. • A manual device – Liquidator requires neither programming nor significant training, so first-time users are performing experiments in a relatively short time. • Inexpensive – unlike automated pipetting stations, the Liquidator 96 is within reach of academic and industrial laboratories.

Liquidator’s many positive attributes make it a powerful tool in many biological and chemical research environments. The following pages demonstrate how genomic and proteomic researchers are using Liquidator to achieve their goals.

2 Liquidator Applications in Genome Research Liquidator 96 has been used in a variety of genomics applications1. Dr. Natalia Ivanova of the Uni- versity of Guelph has found the 200 µL Liquidator very useful for a DNA barcoding workflow that involves DNA extraction from animal/plant material, PCR, sequencing reaction setup, and sequencing product clean-up. Her genomics workflow is detailed below in Figure 1, Applications 1-4. Dr. Ivanova’s workflow requires multiple pipetting steps and the processing of many samples, and the Liquidator provides speed and reproducibility to the overall procedure.

Figure 1: Application 1A DNA Extraction from animal/plant material using Liquidator Application 1: DNA Puriﬁcation

Animal Sample Plant Sample

Tube Rack containing 96-well Plate plant sample containing animal sample New 96-well plate containing 100 µL binding buffer Liquidator 96 adds lysis buffer to wells Liquidator 96 transfers Incubate 50 µL extract to new plate

New 96-well plate

96-well puriﬁcation plate

Liquidator 96 transfers 50 µL of lysed cells to new plate Liquidator 96 transfers 150 µL to puriﬁcation plate

Transfer new plate to robot

Bind to puriﬁcation plate C e n Wash with 180 µL buffer t r i Pure Animal DNA f Wash with 750 µL buffer u g

e Elute in 40-50 µL

Pure Plant DNA 3 Figure 1: Application 2 PCR Amplification Figure 1: Application 3 Cycle Sequencing Reaction Set-up

Application 2: PCR Ampliﬁcation Application 3: Cycle Sequencing Reaction Setup

Animal or Plant DNA Ampliﬁed DNA

New PCR plate with New PCR plate 10.5 µL mastermix with water Liquidator 96 Dilute DNA 1:3 in H2O Liquidator 96 pipettes 2 µL DNA into PCR plate

96-well plate with sequencing reaction components

Place PCR plate in the thermocycler and amplify Pipette 2 µL of diluted DNA into new plate, touch off

Ampliﬁed DNA

Centrifuge and incubate

Primer extension products Unincorporated Dyes

Sequencing Reaction

4 Figure 1: Application 3 Cycle Sequencing Reaction Set-up Figure 1: Application 4 Cycle Sequencing Clean-up

Application 4: Cycle Sequencing Clean-up

Sequencing Reaction

Sephadex G-50 plate

Transfer entire reaction to G-50 plate

Pipette 25 µL of Centrifuge 0.1 mM EDTA

Plate containing 25 µL of 0.1 mM EDTA

Transfer G-50 eluate to new plate

Pure labelled DNA

Seal

Sequence

DNA sequence

5 The 20 µL model is used for research applications at low volume range of 0.5 to 20 µL. Rainin has tested this model for qPCR applications (Figure 2). For both dye and nucleic acid based probe techniques, the Liquidator’s ability to generate consistent qPCR curves with similar Cq values was inves- tigated. The 20 µL Liquidator was used in pipetting 18 µL of qPCR Supermix and 2 µL of diluted DNA template in both cases. The data collected with the Liquidator is 22.24 ± 0.04 for the nucleic acid probe based reactions while the dye based method yielded 10.54 ± 0.04.

The hypothesis tested was that if the Liquidator was able to pipette with 96-channels precisely and consistently, then the Cq values among all 96-channels would nearly identical. If any channels were to fail in pipetting or exhibit inacceptable accuracy, then there would be at least one Cq value that deviated from the majority of the remaining Cq values. Also, if the precision of all of the channels of the LiquidatorqPCR were not Workﬂow optimal for qPCR, then there would be considerable scatter in the Cq data

Liquidator 96 points. There were no pipetting failures observed in this data, and the extremely low uncertainty of the Cq values shows that the device exhibits exquisite precision.

A B

Liquidator 96 pipettes 18 µL of mastermix

Liquidator 96 pipettes C 2 µL of template DNA

Seal plate, mix, centrifuge and place in qPCR machine for analysis

Figure 2. The 20 µL Liquidator 96 Yields Uniform qPCR Data. Panel A shows the overall qPCR workflow carried out using the Liquidator. Panel B shows data obtained using an oligonucleotide probe based assay. The mean Cq value over the 96 channels was 22.24 ± 0.04. Panel C shows data obtained using a dye probe based method. The mean Cq value over 96 channels was 10.54 ± 0.04. 6 Liquidator Applications in Protein Research The Liquidator 96 has been used in a variety of protein chemistry and proteomics research applications (Figure 3). In one example, a paper published in the journal Nature describes how the 200 µL Liquidator was used to set up in cellulo enzymatic assays for a number of evolved enzyme variants2. Another example includes a published study in The Journal of Pediatrics where the 200 µL Liquidator was used to set up an enzymatic MS/MS diagnostic assay for newborns to screen for Fabry’s, Pompe, and mucopolysaccharidosis-I disorders3. General diagrams of how the Liquida- tor was used in these studies are below:

Bacterial cells grown in 96-well plate 110K bloodspots from newborns

Take 3.2 mm punch After 18-22 hour growth at 30° C, and place in 96-well plate protein production is induced for 16-18 hr at 18° C prior to storage at 4° C Liquidator 96 adds 30 µL of enzyme substrates to plate

Plate is incubated 16 hr at 37°C

Liquidator 96 used to resuspend cells Liquidator 96 adds 100 µL of ammonium acetate/ethyl acetate mixture and mixes the samples

Cover with foil and centrifuge

Liquidator is used to transfer 200 µL of the Perform enzyme assay ethlyl acetate phase to a new 96-well plate

Ethyl acetate phase is evaporated under air ﬂow

Liquidator adds 100 µL of mass [Substrate] spectometry mobile phase

Plates are agitated via orbital shaking, covered with aluminum, then placed on injector array for MS/MS

Perform MS/MS

Figure 3. Researcher Uses for the 200 µL Liquidator. Panel A shows the Liquidator 96 used for simple cell resuspension prior to using the intact cells in enzymatic reactions. Panel B shows the uses of the Liquidator in an enzymatic MS/MS detection assay for Fabry, Pompe, and mucopolysaccharidosis-I disorders.

7 Liquidator 96 has also been tested in ELISA applications in comparison to a multichannel pipette4. In this experiment, eight replicate ELISA standard curve samples were set up in two 96-well plates. After briefly incubating the plates at room temperature, all subsequent steps were carried out with an 8-channel multichannel pipette, or the Liquidator 96. The overall protocol for ELISA included wash steps, antibody addition steps, an enzyme substrate addition step, and a quench step (Figure 4). For quantification, plates were read in a multi-well plate reader. The absorbance values were averaged and plotted against initial antigen concentrations.

Plate with 8 replicate standard samples Liquidator 96 Liquidator washes plate 2X with 200 µL PBST buffer

Liquidator adds pimary antibody to wells in 50 µL prior to 5 min incubation

Liquidator washes plate 2X with 200 µL PBST buffer

Liquidator adds horseradish peroxidase conjugated secondary antibody in 50 µL

Liquidator washes plate 3X with 200 µL PBST buffer

Liquidator adds 50 µL of horseradish peroxidase substrate TMB prior to 5 min incubation

Liquidator teminates enzymatic reaction by adding

20 µL of 180 mM H2SO4

Plate is analysed using a plate reader, data is processed and plotted

Figure 4. ELISA Workflow shown for 200 µL Liquidator.

8 The standard curve obtained by carrying out the ELISA experiment using a multichannel pipette was linear over low antigen concentration values, but deviated from linearity at high antigen concentrations (Figure 5A). This is likely due to the inability to wash and incubate samples in each column of the 96-well plate for an equivalent amount of time. The standard curve obtained by using the Liquidator 96 in all ELISA steps is shown in Figure 5B. The standard curve is linear at both low and high antigen concentration values. The R2 of this standard curve is greater than that obtained with the multichannel pipette. The higher quality of the standard curve is probably due to Liquidator’s ability to deliver wash buffers and other liquids to the 96-well ELISA plate in parallel. This particular Liquidator 96 feature allows each sample in the plate to be treated equivalently.

Figure 5. Parallel Pipetting with Liquidator 96 Yields Better ELISA Data. Panel A shows averaged data of eight standard curves obtained using a multichannel pipette. Notice the deviation from linearity for the point beyond 32 ng/µL antigen. Panel B shows averaged data of eight standard curves obtained using the 200 µL Liquidator 96. This fitted line is of higher quality compared to when a multichannel pipette is used to generate the standard curves. The R2 of the standard curve generated with the Liquidator 96 is greater than the standard curve generated using a multichannel pipette. 9 Perspective The 20 µL and 200 µL Liquidator models offer many benefits to biological and chemical researchers carrying out medium and low-volume applications. First, both Liquidator models offer high accuracy and precision. And because it functions similarly to a manual handheld pipette, it is simple to use. For genome research experiments, it can be easily seen that the Liquidator 96 instruments can be applied to PCR, qPCR, nucleic acid extraction, cycle sequencing reaction setup and cycle sequencing probe clean-up. In terms of protein research, the Liquidator can be used for techniques like enzymatic assays, mass spectrometry assays and ELISA.

Liquidator 96 excels in applications where numerous samples require similar treatment. With its 96-channels, Liquidator is more capable of rapidly processing multiple samples than single, 8-channel and 12-channel pipettes. Additionally, because all 96-channels pipette in parallel and with very

Liquidator 96 high reproducibility, time-sensitive experiments are easier to execute and yield better data than when using single- and multi-channel pipettes (i.e., reactions can be started and stopped at the same time and it is impossible to skip or repeat wells and rows in a 96-well plate). Finally, as a manual device with a relative small footprint, the Liquidator 96 easily adapts to complex and varied workflows, whether they be on the lab bench, in a fume hood or cold room, or even in the field.

10 Technical Specifications

Instrument 20 µL Liquidator 96 200 µL Liquidator 96 Volume Range 0.5-20 µL 5-200 µL 20 µL: ± 1.0 % 200 µL: ± 1.0 % 10 µL: ± 1.2 % 100 µL: ± 1.0 % Accuracy 2 µL: ± 6.0 % 20 µL: ± 2.0 % 1 µL: ± 12.0 % 5 µL: ± 5.0 % 20 µL: ≤ 0.8 % 200 µL: ≤ 0.5 % 10 µL: ≤ 1.0 % 100 µL: ≤ 0.8 % Precision 2 µL: ≤ 5.0 % 20 µL: ≤ 1.5 % 1 µL: ≤ 10.0 % 5 µL: ≤ 3.5 % Volume Increment 0.02 µL 0.2 µL Compatible Formats 96 and 384 wells 96 and 384 wells Plate Positions 4 4 Pipetting Technology Air displacement Air displacement Power Requirements n/a n/a Dimensions Both models: 37 cm x 46 cm x 41 cm (w x d x h) (14.5 in x 18 in x 16 in)

11 References 1. Rainin Instrument LLC (2012) CCDB Simplifies Genomic Workflow: Liquidator 96 used in DNA Barcoding. METTLER TOLEDO document. Pages 1-8. 2. Blomberg R, Kries H, Pinkas DM, Mittl PR, Grütter MG, Privett HK, Mayo SL, Hilvert D (2013) Precision is Essential for Efficient Catalysis in an Evolved Kemp Eliminase. Nature 503(7476):418–421. 3. Scott CR, Elliott S, Buroker N, Thomas LI, Keutzer J, Glass M, Gelb, MH, Turecek F (2013) Identification of Infants at Risk for Developing Fabry, Pompe, or Mucopolysaccharidosis-I from Newborn Blood Spots by Tandem Mass Spectrometry. J Pediatr 163(2):498-503. 4. Rainin Instrument LLC (2012) Improving Quantitative ELISA Data: Some Benefits of Using Liquidator 96. METTLER TOLEDO document. Pages 1-4.

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